Liquid metal-heating apparatus for biological/chemical sample

ABSTRACT

An apparatus for temperature control of biological/chemical samples employing liquid metal is described. A gallium-indium alloy may be used to provide excellent temperature control. Methods of using liquid metal to provide temperature control for biological/chemical samples are also described. A preferred use for the described liquid metal-heating apparatus is to provide precise temperature control for polymerase chain reaction (PCR).

This application claims the benefit of Provisional application Ser. No.60/134,264, filed May 14, 1999, and Provisional application Ser. No.60/145,875, filed Jul. 27, 1999.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a heating apparatus forbiological/chemical samples, which device includes PCR thermal cyclers.The present invention relates also to a method of heatingbiological/chemical samples, which method includes PCR.

2. Description of the Related Art

The polymerase chain reaction (PCR) has become a widely used tool inmolecular biology. This technique allows one to quickly and easilyamplify segments of nucleic acid for further investigation and analysis.Roughly two types of automated PCR instruments are conventionallyavailable on the market.

The first type is based on a robotic arm (such as RoboCycler™ fromStratagene). In this type, temperature control is accomplished by usinga stationary heating block, and samples are transferred mechanicallybetween blocks set at different temperatures according to programmedsteps. The samples are moved by the robotic arm in either a circular orlinear direction. The heating blocks comprise wells in which test tubes(or micro titer plate) are fitted, and it is normally necessary to fillthe wells with either water or mineral oil for sufficient heat transfer.

The second type is a fully integrated and dedicated PCR thermocycler.The PCR thermocycler relies on a thermoelectric element for the Peltiereffect to provide rapid change of temperature. Depending on thedirection of electric current, the thermoelectric element can eitherheat or cool a sample on demand. Thermo-cycling parameters areprogrammed into a temperature controller.

The first type uses at least three heating blocks whose temperatures areset at 55° C., 72° C., and 94° C., respectively. The second type uses athermocycler with a heating block whose temperature is controlled tochange to 55° C., 72° C., and 94° C. in one cycle. Conventional heatingblocks have a plurality of wells for receiving test tubes, and theheating blocks heat the test tubes with an electric resistance, forexample, and cool them by circulating a liquid through elaboratechannels inside the heating blocks or by a thermoelectric element, forexample. The fluid for cooling is commonly a water-based medium. Theelaborate channels for cooling are machined into the holding blocks toallow either tap water or refrigerated water to circulate throughout.Although such a setup can give very high cooling rates, high costs areassociated with this system and make this type of configurationunacceptable. Thus, a combination of an electric resistance and athermoelectric element provided in a metal heating block is the mostfrequently used configuration.

The holding block is specifically machined to fit a particular brand oftest tubes in order to provide a maximized contacting surface to enhanceheat transfer. The holding blocks are made interchangeable toaccommodate an assortment of different test tubes or microtiter platesfrom different vendors. Even if the surface of the holding block isprecisely machined, the area actually contacting each sample holder(i.e., test tube or microtiter plate) may vary due to minorimperfections in plastic injection molding. A variety of methods areemployed to alleviate this problem, including force clamping and addingmineral oil, to fill the gap between the surface of a holding block andthe surface of a sample holder. As the number of samples subjected tothe holding block increases, means to ensure temperature uniformitybetween different samples become important.

Further, the heating block itself has temperature distribution. If theheating block has 96 wells, wells at different corners may havedifferent temperatures.

Although heat transfer difference may occur at contacting surfacesbetween test tubes and respective wells, and uneven heat diffusion mayoccur within the heating block, there is no way to verify the accuracyof the temperature. Users must rely on a temperature indicator installedin the heating block.

In addition, the wells of the heating block are designed specificallyfor particular test tubes, and thus, a 96-well format heating blockcannot be used for any other format PCR plates such as a 384-wellformat. Further, one heating block holds only one PCR plate.

SUMMARY OF THE INVENTION

To guarantee the success of experiments and allow users to directlycompare samples from the same run or different runs at different timesusing the same program, it is essential to have all samples reach thesame temperature during each cycle. The uniformity of heating andcooling rates across the entire holding block surface, as well as thephysical fit between wells and test tubes, are very important.

In accordance with the present invention, an improved polymerase chainreaction thermal cycler can be implemented based on liquid metals. Theinvention is based on the realization that liquid metals have acomparable heat conductivity and capacity to that of metal and at thesame time are not confined to having a pre-defined shape. This enablesthe use of sample test tubes from different vendors without switchingtest tube holding blocks. Precise temperature control and rapidtemperature cycling is carried out by liquid metals. In addition,pumping and switching of liquid metal can be based onmagnetohydrodynamics. Furthermore, in one embodiment, multiple platescan be treated at one time when using a large liquid metal bath. Byusing a large liquid metal bath comprising a plurality of heating andcooling sections, which bath has a length sufficient to complete PCRcycles without physically circulating test tubes in the bath, continuousinput of samples and continuous output of PCR products can be performedsimultaneously, resulting in surprisingly high productivity.

The present invention can be adapted to any type of heating and coolingdevice for biological/chemical samples which require accuratetemperature control. The claimed invention is directed to an apparatusfor temperature control of samples comprising at least one containercontaining liquid metal, said container having an upper open area wherethe liquid metal thermally contacts one or more of the samples fortemperature control thereof; and a temperature control device forheating the liquid metal, whereby said liquid metal remains in a liquidstate and does not significantly evaporate during heating.

The container containing the liquid metal may be either a plastic ormetal container. The temperature control device for heating the liquidmetal may be the heat block of a thermal cycler.

A variety of liquid metal compositions may be used in the practice ofthe claimed invention. Compositions containing gallium may be preferred.A most preferred composition may be a 75.5% gallium/24.5% indium alloy.

The apparatus of the presently claimed invention may include a pluralityof containers containing liquid metal and one sample container. Thesample container may then be moved through a series of containerscontaining liquid metal by any convenient means such as manually ormechanically, for example, by use of a robotic arm.

Alternatively, the liquid metal may be moved through the samplecontainer. The liquid metal at a first temperature may be replaced byliquid metal of a second temperature. Movement of the liquid metal,either within the sample container or its injection and removal from thesample container may be accomplished by a conventional pump.Alternatively, movement of the liquid metal may be accomplished bymagnetohydrodynamics in either AC or DC mode. Of course, gravity mayalso be used to move the liquid metal.

The containers containing liquid metal may also be linked to otherapparatus for sample treatment such as a robotic liquid handler anddispenser, a cell incubator and/or a detection system such as a Luminex100, for example.

The claimed apparatus may be used in a method of incubating one or morebiological/chemical samples at a pre-determined temperature comprisingcontacting the one or more biological/chemical samples with a containercontaining liquid metal at the pre-determined temperature for a giventime period. This method may further comprise movement ofbiological/chemical samples between a plurality of containers. Thismovement may be accomplished mechanically by use of a robotic arm, forexample, or manually. In one embodiment, the temperature of theplurality of containers containing liquid metal are 30-65° C., 65-85° C.and 85-100° C., respectively.

The claimed invention also encompasses a method of varying thetemperature of a sample in a single container comprising:

(a) incubating a biological/chemical sample in contact with a containercontaining liquid metal at a pre-determined temperature for a given timeperiod;

(b) changing the temperature of the liquid metal to a pre-determinedtemperature which is different from the predetermined temperature ofstep (a); and

(c) repeating steps (a) and (b) until all desired incubations haveoccurred.

Various means for varying the temperature of the single sample containermay be used. The temperature change may be affected by replacing liquidmetal at a pre-determined temperature with liquid metal at a differentpre-determined temperature. The liquid metal may be moved throughout thecontainer by magnetohydrodynamics. Magnetohydrodynamics may be operatedin either AC or DC mode. Alternatively, the liquid metal may be movedthroughout the container by means of a pump or gravity may be used tomove the liquid metal.

The liquid metal composition of the claimed method may be gallium or acomposition containing gallium. A preferred composition may comprise75.5% gallium and 24.5% indium.

A preferred method using the apparatus of the present disclosure may bea method of performing polymerase chain reaction (PCR). Typically, a PCRcycle comprises:

denaturing a polynucleotide sample in thermal contact with liquid metalat a temperature of about 90-98° C. for about 10-90 seconds;

hybridizing oligonucleotide primers to the denatured polynucleotidetemplate in thermal contact with liquid metal at a temperature of about30-65° C. for about 1-2 minutes; and

synthesizing a new polynucleotide strand incorporating theoligonucleotide primer and using the denatured polynucleotide astemplate for a polymerase in thermal contact with liquid metal at atemperature of about 70-75° C. for about 30 seconds to 5 minutes.

For purposes of summarizing the invention and the advantages achievedover the prior art, certain objects and advantages of the invention havebeen described above. Of course, it is to be understood that notnecessarily all such objects or advantages may be achieved in accordancewith any particular embodiment of the invention. Thus, for example,those skilled in the art will recognize that the invention may beembodied or carried out in a manner that achieves or optimizes oneadvantage or group of advantages as taught herein without necessarilyachieving other objects or advantages as may be taught or suggestedherein.

Further aspects, features and advantages of this invention will becomeapparent from the detailed description of the preferred embodimentswhich follow, although the present invention is not limited thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of this invention will now be described withreference to the drawings of preferred embodiments which are intended toillustrate and not to limit the invention.

FIG. 1 is a schematic side view showing an embodiment of the apparatusof the present invention, in which a single set of samples is connectedto three separate containers containing liquid metal.

FIG. 2 illustrates magnetohydrodynamics using DC mode. FIG. 2(a) is aschematic side view, and FIG. 2(b) is a schematic plane view (showingthe directions of a magnetic field and a electrical field).

FIG. 3 illustrates magnetohydrodynamics using AC mode. FIG. 3(a) is aschematic plane view, and FIG. 3(b) is a schematic side view (showingthe directions of a magnetic field and a electrical field).

FIG. 4 shows connection of multiple reservoirs containing liquid metalwith a KOH reservoir to prevent liquid metal oxidation.

FIG. 5 is a schematic view showing an embodiment with a sloped ramp tofacilitate movement of liquid metal around biological/chemical samples.

FIG. 6 is a schematic side view showing a liquid metal universal adapterfor any microplate or well strip.

FIG. 7 shows a system for processing multiple samples using PCR and theliquid metal baths of the present disclosure.

FIG. 8 is a schematic view showing an embodiment of a PCR continuousflow system using a liquid metal.

FIG. 9 shows a system for continuously processing multiple samples usingPCR and the liquid metal baths of the present disclosure.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Liquid Metal

By using liquid metal as a heating and cooling medium for a heatingblock, full contact with test tubes can be achieved, resulting inuniform heat transfer regardless of the type, size, and shape of testtubes. Pre-formed wells are no longer required. Furthermore, uniformityof temperature within the heating block can be achieved, because liquidmetal can easily be circulated in the heating block by convection or byexternal force.

In this regard, one advantage of using liquid metal is that formation offlow is easily achieved. When liquid metal is exposed to a magneticfield formed in one direction and an electrical field formed in adirection perpendicular to the direction of the magnetic field, theliquid metal flows in a direction perpendicular to both the direction ofthe magnetic field and the direction of the electrical field. By using amagnetic field and an electrical field, it is possible to cause liquidmetal to flow in a designated direction without physical control such ascontrol by a pump. These characteristics can be used to make thetemperature within the heating block uniform by convection or tointroduce and discharge liquid metal into and from the heating block. Inan alternate embodiment a pump may be used. If the electrical field isformed by DC power, and its frequencies change, flow of the liquid metalrapidly oscillates in accordance with the frequencies, thereby achievinghigh uniformity of temperature.

Liquid metal is flowable during operation and has an evaporation pointhigher than operation temperature. Further, liquid metal is preferablynon-toxic under conditions of operation. Liquid metal has high heat andelectrical conductivity and thus can be very responsive to heating andcooling patterns/cycles. For PCR, rapid heating and cooling rates arerequired (such as 4-20° C. per second), which liquid metal can satisfy.

Liquid metal is available on the market for very specific and exclusiveuses, i.e., as a coolant for nuclear power plants where top levelcontrol management is required for safety. Liquid metal is not somethingbiologists normally consider. However, the present inventors discoveredthat some liquid metals could be very useful as heating blocks withoutthe issue of toxicity and other problems. A well-known liquid metal ismercury. However, mercury is not usable in the present invention,because it partially vaporizes at room temperature and is highly toxic.

A variety of liquid metal compositions may be used in the practice ofthe claimed invention. Compositions containing 60-100% gallium incombination with indium may be preferred. Some compositions also containtin and zinc. Some specific examples include: 61% gallium/25.0%indium/13.0% Sn/ 1.0% Zn; 62.5% gallium/21.5% indium/16.0% Sn; 75.5%gallium/24.5% indium; 95% gallium/5% indium; and 100% gallium. In apreferred embodiment the liquid metal may be a gallium-indium alloycomprising 75.5% gallium and 24.5% indium. This alloy becomes a liquidat a temperature of 15.7° C. and it has a boiling point of 2,000° C.Thus, the gallium-indium alloy is in a liquid state but never evaporatesduring PCR. No toxic problem occurs. This alloy is safe even if it isingested. Furthermore, this liquid metal can be washed off easily withdilute potassium hydroxide (KOH) even if the liquid metal adheres totest tubes and other containers. An effective concentration range is0.001M-1M KOH. It does not easily adhere to skin.

In addition to heating blocks for polymerase chain reaction (PCR), theliquid metal heating blocks of the present invention can be used widelyin the field of biotechnology and chemistry. Examples include but arenot limited to incubations of enzymatic reactions such as restrictionenzymes, biochemical assays and polymerase reactions; cell culturing andtransformation; hybridization; and any treatment requiring precisetemperature control. Based on the present disclosure, one of ordinaryskill in the art can readily adapt the liquid metal technology tovarious analyses of biological/chemical samples which require accuratetemperature control.

In a preferred embodiment, the liquid metal heating blocks of thepresently claimed invention may be used for PCR. In a typical PCR cycle,the polynucleotide sample is denatured by treatment in a liquid metalbath at about 90-98° C. for 10-90 seconds. The denatured polynucleotideis then hybridized to an oligonucleotide primer by treatment in a liquidmetal bath at a temperature of about 30-65° C. for 1-2 minutes. Chainextension then occurs by the action of a polymerase on thepolynucleotide annealed to the oligonucleotide primer. This reactionoccurs at a temperature of about 70-75° C. for 30 seconds to 5 minutesin the liquid metal bath. Any desired number of PCR cycles may becarried out.

In a most preferred embodiment, the denaturation of the polynucleotidemay occur at a temperature of 94° C. for about 1 minute. Thehybridization of the oligonucleotide to the denatured polynucleotideoccurs at a temperature of about 37-65° C. for about one minute. Thepolymerase reaction is carried out for about one minute at about 72° C.All reactions are carried out in the same multiwell plate in a liquidmetal bath of the claimed invention. About 30 PCR cycles may bepreferred. The above temperature ranges and the other numbers are notintended to limit the scope of the invention. These ranges are dependanton other factors such as the type of enzyme, the type of container orplate, the type of biological sample, the size of samples, etc. One ofordinary skill in the art can readily modify the ranges as necessary.

Several embodiments included in but not limiting the present inventionwill be explained below.

Liquid Metal Magnetohydrodynamic PCR Thermal Cycler

In one embodiment of the present invention (FIG. 1), a thermal cyclerincludes a sample 2 and a sample container 1 and more than one containerholding the liquid metal 3, 4, 5. In an embodiment, it is preferred tohave the containers made out of plastic materials to prevent chemicalreaction with liquid metal. The liquid metal inside each container isheated by a heating element to a specific temperature according to theneed to complete PCR. Heating the sample to a specific temperature canbe conducted by simply circulating the liquid metal in a loop passing aheating area where the test tubes or micro titer plate is placed 6. Thetest tubes or microtiter plates thermally contact the liquid metal. Thismay be accomplished by contacting the test tubes or microtiter plateswith a container such as a plastic bag containing the liquid metal, forexample. More preferably, in another embodiment, the test tubes ormicrotiter plates are in direct contact with the liquid metal. Changingthe temperature of the heating area can be conducted easily bychanneling each loop through the heating area by use of a valve 7. Theliquid metal can flow by using magnetohydrodynamic force created by amagnetic field and an electrical field whose directions areperpendicular to each other.

Pumping and switching of liquid metal flow is accomplished bymagnetohydrodynamic. Two different modes of magnetohydrodynamic (MHD)flows, DC (direct current) and AC (alternating current), can be used. DCmode MHD is by far the simplest to implement (FIG. 2). It includes apair of electrodes 8 contacting the liquid metal completing anelectrical circuit and a magnetic field. The direction of electricalcurrent flow, the magnetic field, and the flow of liquid metal should bemutually orthogonal to each other. When the electrical current is passedthrough the liquid metal, a magnetic field is created. This magneticfield would have a different orientation with respect to the externalmagnetic field. Therefore, the liquid metal will be pushed along thechannel. No mechanical moving parts are required. DC mode MHD pumping isinherent. However, it requires the electrodes to come in contact withliquid metal. Another variation of MHD is AC mode (FIG. 3). It includesan inductor array 9 and an electronic controller. The electroniccontroller sets off a traveling magnetic wave in the inductor array. Thetraveling magnetic field wave introduces a current inside the liquidmetal. The same current will push the liquid metal inside the channel inthe same direction as the traveling magnetic field. In AC MHD, there isno need to have electrodes directly contacting the liquid metal itself.However, it is generally a bit more difficult to predict the performanceof the pump. A switch mechanism is needed to divert the flow of liquidmetal back to its corresponding temperature reservoir. This can beaccomplished by using either DC or AC mode MHD. It simply deflects theflow of liquid metal from its gravity flow path.

Over time, the flow switch may accumulate into one particular reservoirmore than another. Although this is not a problem in terms oftemperature control, the volume of liquid metal under differenttemperatures would be different. To overcome this problem, a smalltubing 10 is used to connect different reservoirs together (FIG. 4). Thesmall tubing allows the liquid metal level to equilibrate, yet it doesnot significantly affect the temperature of each individual liquid metalreservoir.

To prevent liquid metal from degrading due to oxidation in theatmosphere, a light KOH solution is needed. Again, a small tubing 11connects a KOH reservoir with different liquid metal reservoirs. The KOHsolution should be cooled by means of a thermoelectric element to 4Cafter the thermal cycle is finished to preserve the sample. KOH solutioncan be pumped by either MHD or a conventional fluid handling pump. KOHsolution is also conductive.

In an alternate embodiment, the test tubes 12 or micro titer plate areplaced on a slightly sloped ramp 13 with openings in the upper and lowerends (FIG. 5). Once the liquid metal is pumped out of the upper opening,it will flow naturally by gravity towards the lower opening. The upperopening should allow flow of liquid metal covering the entire upper rampsurface. In normal embodiments, the depth of the liquid metal flow issuch that it covers the depth of the sample inside the test tube ormicro titer plate. In an embodiment, it is preferred to have a loweropening to collect the liquid metal to a small opening to be drainedback to its corresponding reservoir 14 to be re-heated.

Universal Heating Block with Liquid Metal

One problem with thermal cyclers currently available is the uniquephysical design of the heat blocks of many of the thermal cyclerscurrently available. Because the use of a thermal cycler requires theuse of consumable plastic microplates and/or trips of wells in which thereactions are performed, many manufacturers have chosen to uniquelyconfigure their heat blocks thereby forcing a user to purchase their ownbrand of plastic consumables. That is, “generic” plastics commerciallyavailable from other sources won't fit onto the heat block. Thisrestricts the freedom a researcher has in that he/she is forced topurchase plastics from only the manufacturer of his/her thermal cycler.A solution to this limitation would provide flexibility to theresearcher in allowing a greater choice of plastics.

A second embodiment of the claimed invention presents a solution to theproblem stated above, while maintaining a high level of temperaturecontrol. A universal adapter for any plastic microplate (including96-well, 384-well and 1536-well) and strips of wells (including 8-well,12-well or any portions or combinations thereof) functions on anycommercially available thermal cycler (or equivalent). A liquid metalalloy bath is constructed from a container made of metal (brass, copperor other similar metals or combinations thereof) or plastic. A volume ofthe liquid metal alloy is then placed into the metal or plasticcontainer (FIG. 6). This bath is placed in contact with the heat blockof a conventional thermal cycler 15. The plastic part used for thereaction is placed into the liquid metal alloy bath 16. Caps and/orsuitable cover are placed onto the plastic part to prevent evaporationduring the run. The thermal cycler is then programmed and run as normalwith the cover open. Given the excellent thermal conductivity propertiesof the liquid metal alloy bath, efficient, reliable, and reproducibletemperature control is maintained throughout the run. In a preferredembodiment, a gallium-indium alloy may be used as the liquid metal. Thegallium-indium alloy is a solid at 4° C. Since most thermal cycler runsare programmed to end at 4° C., the alloy freezes and allows very easyremoval of the plastic part from the bath without any appreciable alloyadhering to the surface of the plastic.

Other advantages of the liquid metal alloy bath are that the lag timewhen changing temperature of a water bath is virtually eliminated. Theliquid metal alloy bath provides excellent heat conductivity withminimal ramp times between different temperatures. Furthermore, there isno evaporation of the alloy, thus eliminating most user intervention andmaintenance.

The liquid metal alloy bath may be designed to use on any commerciallyavailable heat block, including thermal cycler heat blocks, using anycommercially available plastic consumables such as those designed forPCR.

In an alternate embodiment, the liquid metal alloy baths may bevertically stacked with heating elements sandwiched between the baths.This multi-bath configuration allows multiple consumables to be used forPCR simultaneously rather than sequentially. This approach provides aneffective solution to satisfy the demands of high throughput and/or highvolume applications.

Liquid Metal Robocycler

In this embodiment, there are a plurality of heating containerscontaining liquid metal and a heating device for each heating containercontaining the liquid metal. The samples are moved either manually ormechanically between the temperature blocks. In a preferred embodiment,the samples may be moved mechanically between different temperatureblocks according to pre-programmed steps. The samples are moved by arobotic arm in either circular or linear movement.

Liquid Metal High Capacity Thermocycler

A plurality of containers containing liquid metal will be employed in anautomated system using a robotic liquid handler and dispenser such asthe Biomek 2000 from Beckman Coulter (FIG. 7). In a preferredembodiment, three containers may be used at temperatures of 30-65° C.,65-85° C. and 85-100° C., respectively. The system will be enclosed in aplastic box-like container with purified air supplied through a HEPAfilter. The system will also include a vacuum manifold for filtration, atemperature control unit and a temperature monitoring system for useduring thermocycling. Using a kit such as the mRNA Express Kit, theworkstation may be used for collection of cells from multi-well plates,cell lysis, mRNA purification, cDNA synthesis, and amplification by PCRwithout human intervention. After PCR, the GenePlate from the mRNAExpress Kit is automatically heated at 94° C. and the solutions fromeach well are transferred to fresh microplates where captureoligonucleotide-immobilized Luminex beads and streptavidin-PE (dye) werealiquoted previously. The GenePlate is then transferred to the LuminexX-Y station for detection. Although sample preparation and Luminexdetection each have ample and satisfactory throughput, the time limitingfactor is PCR which can take 1-3 hours depending on individualapplications. In one embodiment, the combined throughput of this systemis 96 samples every 1-3 hours. Because 6-10 genes can be analyzedsimultaneously in each well of the GenePlate, a total of 576-960 genescan be analyzed every 1-3 hours.

Ultra-high Throughput Gene Amplification Platform

In the high throughput model, a MegaCycler (Hudson Control) is placednext to the Biomek 2000. In a preferred embodiment, the MegaCycler canaccommodate up to six GenePlates simultaneously and the Beckman CoulterORCA robotic arm (or equivalent) transfers the GenePlates between theBiomek 2000, the MegaCycler, and the Luminex instruments.

In the above, the time limiting factor is PCR which takes 1-3 hours forsufficient amplification. In one embodiment, a PCR factory includes, forexample, 90 liquid metal baths for 30 cycles arranged in a linearassembly-line fashion. Each bath may have a length sufficient toaccommodate one or two PCR plates. The number of liquid metal baths canfreely be selected depending on the intended number of cycles. Because aliquid metal has excellent heat conductivity and heat transfer andallows PCR plates to flow thereon, it is possible to conduct acontinuous flow system. This system allows analyzing 96 samples (576-960genes) every 2 minutes. Surprisingly, this means that the human genomecan be fully sequenced in less than one day. As shown in FIG. 8, PCRplates 81 are coupled in line with a string 82 which continuously movesthe PCR plates in one direction. The PCR plates flows on top of a liquidmetal 83 filling each bath 84 (#1 to #n). The baths 84 are arranged inline and are heated by a heater 85 provided at the bottom of the bath.Any heating method can be employed. Each bath 84 may hold 200cc to1,000cc of the liquid metal 83.

In an embodiment, by using the above continuous flow system,polynucleotide analyses can be conducted by arranging other systemsupstream and downstream of the continuous flow system. FIG. 9 shows anexample. From one end, a GenePlate, which already has cDNA synthesizedfrom captured mRNA, is placed in the first bath, remains for 30-90seconds and is then robotically transferred to the next bath. Thisprotocol continues with each successive liquid metal bath. At the otherend, PCR-completed GenePlates are removed every 30-90 seconds in asimilar assembly line fashion. With this arrangement, the time limitingfactors are the Biomek 2000 and the Luminex instrument. By switching tothe superior Biomek F/X, a GenePlate can now be treated every twominutes. The current Luminex 100 instrument has only one nozzle, andrequires approximately 10-20 minutes of processing time for eachGenePlate. Additional Luminex 100 instruments could be incorporated intothe system. In the above, each device can be of any type or model. Oneof ordinary skill in the art can readily obtain devices as necessary.

Uniformity of Temperature

The following Table 1 demonstrates the uniformity of temperature for theliquid metal bath compared to a commercially available thermocycler anda water bottle. It can be seen from the data that both variation at agiven location in the heating apparatus and variation between locationswithin the heating apparatus are much less for the liquid metal baththan for a conventional thermocycler. For the liquid metal bath,variation in temperatures measured at 5 different locations is 74.5° C.to 73.1° C. or a range of 1.4° C. In contrast, the temperature variationfor a conventional thermocycler measured at 5 locations is 71.3° C. to68.9° C. or a range of 2.4° C. Likewise, the variability of temperatureat any given point in the heating apparatus is greater for theconventional thermocycler than for the liquid metal bath. Variabilitiesas high as +/−3.3° C. are observed with the conventional thermocyclerwhile the greatest variability with the liquid metal bath is +/−0.09° C.Clearly, the liquid metal bath of the presently claimed inventionprovides more precise temperature control compared to temperatureregulators currently available.

Uniformity of temperature of a liquid metal bath can be improved bycirculating the liquid metal in the bath. Circulation of the liquidmetal can be created by natural convection, forced convection using apump or megnetohydrodynamic power, vibration by physical force ormegnetohydrodynamic power with DC current, etc. When using a PCR platesuch as a microtiter plate, even if circulation becomes steady flow inthe bath, the steady flow can easily be unsteady or turbulent whenplacing or submerging the plate in the liquid metal, resulting in auniform temperature distribution.

TABLE 1 Temperature uniformity Location Control* Liquid metal**Thermocycler*** of sensor (n = 7) (n = 7) (n = 6) Sensor upper 21.8 ±0.05° C. 73.1 ± 0.09° C. 70.8 ± 0.97° C. 1 left corner Sensor lower 21.7± 0.09° C. 73.8 ± 0.08° C. 71.0 ± 1.02° C. 2 left corner Sensor upper21.8 ± 0.09° C. 74.0 ± 0.08° C. 71.3 ± 1.11° C. 3 right corner Sensorlower 21.6 ± 0.05° C. 74.5 ± 0.00° C. 71.2 ± 0.83° C. 4 right cornerSensor middle 21.7 ± 0.00° C. 74.2 ± 0.05° C. 68.9 ± 3.33° C. 5 *5sensors are placed in the same water bottle. **5 sensors are placed inthe different locations within the same liquid metal bath ***5 sensorsare placed in the different wells of heat block of commerciallyavailable model 480 thermocycler (PR Bio).

It will be understood by those of skill in the art that numerous andvarious modifications can be made without departing from the spirit ofthe present invention. Therefore, it should be clearly understood thatthe forms of the present invention are illustrative only and are notintended to limit the scope of the present invention.

What is claimed is:
 1. An apparatus for temperature control of samples,comprising: at least one container containing liquid metal, saidcontainer having an upper open area where the liquid metal thermallycontacts one or more of the samples for temperature control thereof; anda temperature control device for heating the liquid metal at least overa temperature range from about 65° C. to about 85° C., whereby saidliquid metal remains in a liquid state and does not significantlyvaporize at a temperature within said range, wherein said metal is in aliquid state throughout said temperature range.
 2. The apparatus ofclaim 1 wherein the container containing liquid metal is placed incontact with the heat block of a thermal cycler.
 3. The apparatus ofclaim 1 wherein the container containing liquid metal is a metalcontainer.
 4. The apparatus of claim 1 wherein the container containingliquid metal is a plastic container.
 5. The apparatus of claim 1 whereinthe liquid metal is a gallium-indium alloy.
 6. The apparatus of claim 5wherein the gallium-indium alloy comprises 75.5% gallium and 24.5%indium.
 7. The apparatus of claim 1 which comprises a plurality ofcontainers containing liquid metal and one sample container.
 8. Theapparatus of claim 7 further comprising a robotic arm for moving thesample containers between the plurality of containers containing liquidmetal.
 9. The apparatus of claim 1 wherein the liquid metal is movedthroughout the container by magnetohydrodynamics.
 10. The apparatus ofclaim 1 wherein the liquid metal is moved throughout the container by apump.
 11. The apparatus of claim 1 wherein gravity is used to move theliquid metal.
 12. The apparatus of claim 1 wherein a plurality ofcontainers containing liquid metal are operably linked to a roboticliquid handler and dispenser.
 13. The apparatus of claim 12 whereinthree containers containing liquid metal are maintained at 30-65° C.,65-85° C. and 85-100° C., respectively.
 14. A method of incubating oneor more biological/chemical samples in one or more containers at apredetermined temperature, comprising contacting the one or morecontainers with liquid metal at the pre-determined temperature for agiven time period, wherein said metal is in a liquid state at least overa temperature range from about 65° C. to about 85° C., and wherein saidliquid metal remains in a liquid state and does not significantlyvaporize at a temperature within said range.
 15. The method of claim 14,wherein the biological/chemical samples in one or more containers aremoved manually to contact the liquid metal.
 16. The method of claim 14,wherein a robotic arm moves the biological/samples in one or morecontainers to contact the liquid metal.
 17. The method of claim 14,wherein the predetermined temperatures for said containers are 30-65°C., 65-85° C. and 85-100° C., respectively.
 18. A method of varying thetemperature of a biological/chemical sample, comprising: (a) incubatinga container containing the biological/chemical sample in liquid metal ata first pre-determined temperature for a given time period; and (b)incubating the container containing the biological/chemical sample inliquid metal at a second pre-determined temperature which is differentfrom the first pre-determined temperature.
 19. The method of claim 18,wherein the temperature change is affected by replacing liquid metal atthe first predetermined temperature with liquid metal at the secondpredetermined temperature.
 20. The method of claim 19, wherein theliquid metal is replaced by moving using magnetohydrodynamics.
 21. Themethod of claim 20, wherein the magnetohydrodynamics is in AC mode. 22.The method of claim 20, wherein the magnetohydrodynamics is in DC mode.23. The method of claim 19, wherein the liquid metal is moved by a pump.24. The method of claim 19 wherein gravity is used to move the liquidmetal.
 25. The method of claim 18 wherein the liquid metal is agallium-indium alloy.
 26. The method of claim 25 wherein thegallium-indium alloy comprises 75.5% gallium and 24.5% indium.
 27. Amethod of carrying out polymerase chain reaction (PCR), wherein one PCRcycle comprises: denaturing a polynucleotide sample in thermal contactwith liquid metal at a temperature of about 90-98° C. for about 10-90seconds; hybridizing oligonucleotide primers to the denaturedpolynucleotide sample in thermal contact with liquid metal at atemperature of about 30-65° C. for about 1-2 minutes; and synthesizing anew polynucleotide strand incorporating the oligonucleotide primer andusing the denatured polynucleotide sample as a template for a polymerasein thermal contact with liquid metal at a temperature of about 70-75° C.for about 30 seconds to 5 minutes.
 28. An apparatus for temperaturecontrol of samples, comprising: at least one container containing liquidmetal, said container having an upper open area where the liquid metalthermally contacts one or more of the samples for temperature controlthereof; and a temperature control device for heating the liquid metal,whereby said liquid metal remains in a liquid state and does notsignificantly evaporate during heating, and wherein said containercontaining liquid metal is a plastic container.
 29. An apparatus fortemperature control of samples, comprising: at least one containercontaining liquid metal, said container having an upper open area wherethe liquid metal thermally contacts one or more of the samples fortemperature control thereof; and a temperature control device forheating the liquid metal whereby said liquid metal remains in a liquidstate and does not significantly evaporate during heating, wherein saidliquid metal is a gallium-indium alloy.
 30. The apparatus of claim 29,wherein the gallium-indium alloy comprises 75.5% gallium and 24.5%indium.
 31. An apparatus for temperature control of samples, comprisinga plurality of containers containing liquid metal and at least onesample container, wherein at least one container containing liquid metalhaving an upper open area where the liquid metal thermally contacts oneor more of the sample containers for temperature control thereof; atemperature control device for heating the liquid metal, whereby saidliquid metal remains in a liquid state and does not significantlyevaporate during heating, and a robotic arm for moving the samplecontainers between the plurality of containers containing liquid metal.32. The apparatus for temperature control of samples, comprising: atleast one container containing liquid metal, said container having anupper open area where the liquid metal thermally contacts one or more ofthe samples for temperature control thereof; and a temperature controldevice for heating the liquid metal, whereby said liquid metal remainsin a liquid state and does not significantly evaporate during heating,wherein the liquid metal is moved throughout the container bymagnetohydrodynamics.
 33. An apparatus for temperature control ofsamples, comprising: at least one container containing liquid metal,said container having an upper open area where the liquid metalthermally contacts one or more of the samples for temperature controlthereof; and a temperature control device for heating the liquid metal,whereby said liquid metal remains in a liquid state and does notsignificantly evaporate during heating, and wherein a plurality ofcontainers containing liquid metal are operably linked to a roboticliquid handler and dispenser.
 34. The apparatus of claim 33, whereinthree containers containing liquid metal are maintained at 30-65° C.,65-85° C. and 85-100° C., respectively.
 35. A method of incubating oneor more biological/chemical samples at a predetermined temperaturecomprising contacting the one or more biological/chemical samples with acontainer containing liquid metal at the predetermined temperature for agiven time period, wherein a robotic arm moves the biological/chemicalsamples between a plurality of containers containing liquid metal. 36.The method of claim 35, wherein the predetermined temperatures for saidplurality of containers are 30-65° C., 65-85° C. and 85-100° C.,respectively.
 37. A method of varying the temperature of a sample in asingle container, comprising: (a) incubating a biological/chemicalsample in contact with a container containing liquid metal at a firstpredetermined temperature for a given time period; (b) changing thetemperature of the liquid metal to a second predetermined temperaturewhich is different from the predetermined temperature of step (a); (c)repeating steps (a) and (b) until all desired incubations have occurred;and wherein the temperature change is affected by replacing liquid metalat the first predetermined temperature with liquid metal at the secondpredetermined temperature, and wherein the liquid metal is movedthroughout the container containing liquid metal bymagnetohydrodynamics.
 38. The method of claim 37, wherein themagnetohydrodynamics is in AC mode.
 39. The method of claim 37, whereinthe magnetohydrodynamics is in DC mode.
 40. A method of varying thetemperature of a sample in a single container, comprising: (a)incubating a biological/chemical sample in contact with a containercontaining liquid metal at a first predetermined temperature for a giventime period; (b) changing the temperature of the liquid metal to asecond predetermined temperature which is different from thepredetermined temperature of step (a); (c) repeating steps (a) and (b)until all desired incubations have occurred; and wherein the liquidmetal is a gallium-indium alloy.
 41. The method of claim 40, wherein thegallium-indium alloy comprises 75.5% gallium and 24.5% indium.
 42. Themethod of claim 18, wherein the incubating at the first pre-determinedtemperature and the incubating at the second pre-determined temperatureare performed in a single liquid metal bath, said method additionallycomprising changing the temperature of the liquid metal in saidcontainer from the first pre-determined temperature to the secondpre-determined temperature between the incubating at the firsttemperature and the incubating at the second temperature.